Reverse transcriptase (RT) is a kind of DNA polymerase encoded by retroviruses, which can synthesize DNA using DNA or RNA as template. Reverse transcriptase, which can convert RNA into cDNA, is widely used in molecular biology, including constructing cDNA libraries and analyzing the amount of RNA in biological samples by RT-PCR. Nowadays the dominant RT in the market is murine leukemia virus reverse transcriptase (MLV-RT).
MLV-RT is composed of two functional domains, a DNA polymerase domain at the N-terminus and a ribonuclease (RNase) H domain at the C-terminus. These two domains can be expressed separately without affecting the function of each other. The first generation of recombinant MLV-RT used in cDNA synthesis contained only the DNA polymerase domain, with the RNase H domain deleted. Though the enzymatic activity is similar to that of full-length MLV-RT, its processivity is relatively poor, resulting in short cDNAs. It was found that although the RNase H domain does not affect the DNA polymerase activity of MLV-RT, it affects the enzyme's processivity. The reason is that the RNase H domain binds to the template/primer complex, and thereby increases the enzyme's affinity for the template/primer. When the 524th Asp, a key residue in the RNase H active site, is replaced with Asn by site-directed mutagensis, the resulting mutant enzyme, MLV-RT-D524N, does not demonstrate any RNase H activity, but retains the DNA polymerase activity and high affinity for the template/primer. This mutant enzyme, which is patented by Invitrogen under trade mark Superscript II, is currently widely used. However, this mutant enzyme is still not perfect; its processivity is still not ideal and the most common problem encountered is the formation of short, less than full-length products.